Type B hepatitis is a viral disease which occurs frequently in particular in the tropics of Africa, in Southeast Asia and in the Far East. Hepatitis B is suggested to be causative of chronic hepatitis or hepatic cirrhosis or, further, primary liver cancer. The pathogen is hepatitis B virus (hereinafter abbreviated as "HBV"), which is one of the DNA viruses. Occurring as a spherical particle having a diameter of 42 nm, it is called "Dane particle" after the discoverer. In the outer layer, there is HBV surface antigen (hereinafter abbreviated as "HBsAg"). It includes subtypes adr, adw, ayr, ayw and so on, which differ in antigenicity. The subtypes adw and adr are common in Japan.
In the blood of patients with hepatitis B, there are also detected smaller particles and tubular particles besides Dane particles. These particles have been shown to contain the same type of HBsAg as found in the Dane particle. For other viruses, it is known that an antibody to the surface antigen of a virus can prevent infection with said virus. In the case of HBV, it may be anticipated that a vaccine against hepatitis B might be produced based on HBsAg. However, human and chimpanzee alone are susceptible to HBV infection. Attempts to infect cultured cells have been unsuccessful. Therefore, the source of HBsAg is limited to the blood of infected human patients. While the small and other particles obtained may meet the demand therefor as a material for preparing diagnostic reagents, the supply is still insufficient for large-scale vaccine production.
Recent advances in molecular biology have made it possible to transform bacteria by introducing thereinto a DNA coding for a nonbacterial protein. If the structural gene for HBsAg (hereinafter abbreviated as "HBsAg gene") can be expressed in bacteria as a result of the use of this gene manipulation technique, it will become possible to produce HBsAg, substantially free from the risk of HBV infection, in large quantities and to open a way to practical use of hepatitis B vaccine.
For the subtype ayw found predominantly in Europe and America among the currently known four subtypes adw, adr, ayw and ayr, the locus and base sequence of the HBsAg gene have been determined [Galibert, F. et al., Nature, 281, 646 (1979); Charnay, P. et al., Nucleic Acids Res., 7, 335 (1979)] and its expression as a hybrid protein in Escherichia coli has been reported [Charnay, P. et al., Nature, 286, 893 (1980); Edman, J. C. et al., Nature, 291, 503 (1981)].
For the subtypes adw and adr, which are frequently found in Japan, some of the present inventors have succeeded in constructing a DNA containing the HBsAg gene, determined the DNA base sequence of said gene and the locus thereof on the genome, and opened a way to large-scale HBsAg production by cultivation of a transformant carrying this recombinant DNA (Japanese Unexamined Patent Publication Nos. 194897/1983, 201796/1983 and 74985/1984).
As recently reported by Machida, A. et al. [Gastroenterology, 85, 268 (1983); ibid., 86, 910 (1984)], the HBsAg small particles obtained from the plasma of hepatitis B virus e antigen-positive patients have been shown to contain a P31 protein (molecular weight: 31 kilodaltons) and a P35 protein (a conjugated protein with a sugar joined to P31; molecular weight: 35 kilodaltons) in addition to those principal peptides so far identified, such as P-I (molecular weight: 22-24 kilodaltons) and P-II (molecular weight: 25-29.5 kilodaltons) [Peterson, D. L. et al., Proc. Natl. Acad. Sci. USA, 74, 1530 (1977)]. P31 is composed of P-I and 55 amino acid residues encoded by the Pre-S region joined to the N-terminus of P-I, and the presence of a polymerized human serum albumin (poly-HSA) receptor in this region has been established. The above-cited report published in 1984 shows that the above P31 protein has a sugar chain. On the other hand, it is considered that the Dane particle adheres to the hepatocyte via poly-HSA for enabling proliferation since poly-HSA and the receptor therefor are present also on the hepatocyte surface. Therefore it is expected that successful masking of the poly-HSA receptor on the Dane particle with an antibody to P31 would prevent said particle from binding to the hepatocyte, hence would contribute to more effective prevention of HBV infection.
On the other hand, restrictive hydrolysis of P31 with various proteases, for example trypsin, cleaves P31 selectively at the site of the 48th amino acid (arginine) residue from the N-terminus, giving a protein (P28) having a molecular weight of 28 kilodaltons. Also in the case of P31 gene expression in Saccharomyces cerevisiae, a tendency is seen toward decomposition of the gene product due to the presence of a large quantity of trypsin-like protease in yeast cells, which leads to formation of P28 (FIG. 1). In addition to this P28, 30-, 21- and 19-kilodalton proteins, among others, may possibly be formed as a result of decomposition of the 16th and 18th arginine residues and the 177th and 196th lysine residues from the N-terminus of P31 in the presence of trypsin-like protease in the yeast.
As mentioned above, the HBsAg particles obtained from the plasma of hepatitis B virus e antigen-positive patients contain a P31 protein and a P35 protein (a conjugated protein with a sugar joined to P31; molecular weight: 35 kilodaltons) in addition to principal peptides such as P-I and P-II [Stibbe, W. et al., J. Virol., 46, 626(1983)]. In particular, the main body of the infections HBV named as Dane particle possesses P31 protein and P35 protein besides P-I and P-II as surface antigens. It would be more effective to use plural antigens found in HBV than to use a single antigen as a vaccine material. That is, a preferable vaccine should have P31 protein and P35 protein as well as P-I and P-II. Although production of vaccine against hepatitis B virus by recombinant DNA techniques has been attempted, only Michel, M. L. et al report [Proc. Natl. Acad. Sci. USA, 81, 7708 (1984); Bio/Technology, 3, 561(1985)] a vaccine material that contains plural antigens found in HBV. However, their methods require the use of an animal cell as a host which suffers the disadvantage of being very costly.